Vascular endothelium regulates smooth muscle tone through the coordinated release of agents such as prostacycline, endothelium- derived relaxing factor (EDRF) or nitric oxide, and endothelin. Recent studies, both in isolated cardiac multicellular preparations and in intact hearts in vivo, indicated that endocardial endothelium may similarly influence myocardial contraction predominantly by modulating the onset of relaxation. Bioassay studies using cultured endocardial endothelial cells and isolated cardiac papillary muscle preparations suggest that diffusible agents are involved, one of which is probably nitric oxide. However, the relative contribution of endocardial endothelial and coronary vascular endothelial cells to these effects, and the myocardial mechanism of action of substances released by these cell types is unclear. We studied the effects of effluent of superfused endocardial and vascular endothelial cultured cells on contraction and intracellular calcium transients of isolated adult rat cardiac myocytes. Both endocardial and vascular endothelial cells tonically released a novel substance which rapidly and reversibly decreased the amplitude of myocytes twitch contraction by inducing earlier relaxation, and also increased diastolic cell length. These effects were not associated with any change in the intracellular calcium transient, indicating cardiac myofilament "desensitization". The activity of endothelial cell effluent remained stable at 37 degrees C for several hours or at 4 degrees C for at least 48 hours. The action of this substance did not involve nitric oxide, cyclic GMP or prostanoids, nor changes in intracellular pH. These properties suggest that endothelial cells may rapidly modulate cardiac contraction- relaxation coupling and diastolic tonus by altering myofilament properties, as well as exert distant effects because of the unusual stability of this substance